Coeliac disease is a condition in which the lining of the small intestine is damaged by gluten, a mixture of different storage proteins found in the starchy endosperm of wheat and rye grains as well as in closely related species. The gluten matrix consists of approximately equal mixtures of gliadin and glutenin proteins. Coeliac disease is primarily caused by the gliadin proteins. Specifically, in this disease the villi of the small intestine are destroyed and the lining becomes flattened, seriously impairing nutrient absorption. Typical symptoms are weight loss, foul-smelling diarrhoe, vomiting, abdominal pain and swelling of the legs. The only cure currently available is a life-long gluten-free diet strictly avoiding all food and pharmaceutical compositions containing wheat, rye and barley.
Further, a number of humans suffer from general intolerance to glutens. The range of intolerance varies greatly although there are no clear clinical symptoms as in coeliac disease. Many humans therefore benefit from reducing the uptake of gluten.
When wheat flour is mixed with water the gluten proteins develop a unique, viscoelastic structure which enables the preparation of a large number of food products. Flour mixed with a small amount of water develops a dough, from which leavened bread, unleavened bread (for example nan, chapattis), biscuits, cookies, and cakes are made in various parts of the world. When a larger amount of water is added to flour, batters are formed and these are used to make wafers, pancakes and other products. Doughs made from durum wheat are used to make cous cous and pasta products: spaghetti, macaroni and the like. In all of these products the gluten matrix traps gas formed during cooking, which allows the developing product to rise/extend through viscous flow and yet maintains the structure (of the product) by developing elastic restraint.
The gliadin proteins comprise a complex mixture of monomeric polypeptides ranging from about 30,000 to 70,000 in molecular weight. The gliadins are often classified into distinct groups, the α/β-, γ- and ω-gliadins.
In contrast, the glutenin proteins have a molecular weight of up to several millions and are among the largest natural polymers known today. They are formed from polypeptide subunits linked via disulphide bonds. The majority of the monomers have a molecular weight which is similar to the gliadins and are called low-molecular-weight (LMW) subunits. The remainder are larger and are referred to as high-molecular-weight (HMW) glutenin subunits. Gliadins and glutenins are conveniently separated by extracting flour with aqueous alcohols, as the gliadins are soluble and the glutenins insoluble under these conditions.
The gliadins impart viscous flow or extensibility to a dough whereas glutenins impart both elasticity and extensibility. The large glutenin molecules contribute more to elasticity and less to extensibility, whereas the smaller glutenin molecules have the reverse effect. Thus the mean molecular weight distribution of the glutenin monomers as well as the ratio of glutenin to gliadin governs the visco-elasticity of the dough. The different foodstuffs of wheat described above require different visco-elastic properties. For example bread requires a balance of extensibility (to allow the dough to rise) and elasticity (to hold the dough volume before it sets in the oven) whereas biscuits require maximum extension (to enable thin dough sheets to be produced) and minimal elasticity (which would cause biscuit shrinkage prior to baking).
The gluten proteins of wheat are also consumed in a number of other products. Isolated gliadins have film-forming properties and are used as a surface layer or coating on confectionery to inhibit stickiness. Gliadins are further applied as a surface coating to pharmaceutical tablets for the same reason, or to inhibit the taste of unpleasant flavours during consumption of the tablet. Individuals with severe forms of coeliac disease will already be at risk from consumption of these products.
The structures of α/β-gliadins, γ-gliadins and LMW glutenin subunits are closely related and amino acid sequences are homologous in two of five sequence domains (see FIG. 1).
Nearly all LMW genes have 5 domains. The first domain containing one cystein residue is missing only in two sequences analyzed so far. Domain II, so-called repetitive domain is highly variable among the different members of LMW subfamilies. Domain III is a highly conserved domain containing 5 cystein residues found in all LMW genes. Domain IV is glutamine rich, but variable between the different subfamilies. Domain V is a conserved sequence terminating the LMWs and containing the sixth and final cystein residue. The derived amino acid sequences would result in polypeptides ranging from 33.4 to 39.5 KDa.
α- and γ-gliadins and LMW glutenins contain 6 or 8 cysteine residues that form 3 or 4 homologous intramolecular disulfide bonds. The location of these cysteine residues in the amino acid sequence is conserved among most LMW glutenin proteins. Two of the cysteine residues present in LMW glutenins form intermolecular disulfide bonds with the cysteine residues of other glutenin proteins. These 2 cysteine residues are thus responsible for the aggregative nature of LMW glutenin subunits. The structure of the storage proteins and the relevance of the cysteine residues for intermolecular crosslinking of glutenins has been analyzed in great detail in the art (Weegels and Hamer, J. Cereal Science, vol. 25 (1997), 155-163; Orsy et al., J. Biological Chemistry, vol. 276 (2001), 32322-32329; Shewry and Tatham, J. Cereal Science, vol. 25 (1997), 207-227; Lindsay et al., J. Cereal Science, vol. 31 (2000), 321-333; and Müller et al., J. Cereal Science, vol. 27 (1998), 109-116).
The use of genetic engineering methods for modifying or improving wheat gluten and wheat products has been suggested in the art. Vasil and Andersen for example suggested to modify wheat gluten by increasing the numbers of HMW-GS genes, to alter the number and position of cysteine residues, to change the physical characteristics of the gluten matrix, to change the composition and arrangement of the repeat motifs in the repetitive domain, to increase the proportion of glutenins in the large polymer fraction, to modify the polymer network, to introduce LMW-GSs with only one available cysteine for intermolecular bonds as polymer chain terminators and to modify the LMW-GSs to achieve modification of wheat quality (Trends Plant Sci., vol. 2 (1997), 292-297).
Modified glutenin genes, wherein sequences encoding a domain which confers the ability to incorporate into a gluten or bind a ligand or other macromolecule were introduced into the gene sequence, have further been reported in the art (WO00/02914).
However, so far no suggestions have been made for improving the gluten composition in such a manner that foodstuffs prepared thereof are less of a risk to patients with coeliac disease and/or other forms of gluten intolerance.
The present invention thus seeks to provide gliadin-free or gliadin-reduced foodstuff prepared from wheat, maize and/or flour derived thereof, beneficial for feeding patients suffering from coeliac disease and/or other forms of gluten intolerance, while maintaining the advantagous functional characteristics of conventional wheat, maize and/or flour containing for example wheat storage proteins.